Abstract
Background: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs with multiple biological functions. CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2 (CNOT2), which was found to regulate tumor cell apoptosis through caspase pathway. Methods: Potential circRNA.0007127 target microRNAs (miRNAs) were analyzed by miRanda, TargetScan, and RNAhybrid software, and the miRNAs with binding sites for apoptosis-related genes were screened. The roles of circRNA.0007127 and its downstream target, microRNA (miR)-513a-5p, were validated by quantitative real-time polymerase chain reaction (qPCR), flow cytometry, mitochondrial membrane potential, immunofluorescence, western blot, and caspase-8 (CASP8) protein activity in vitro in H2O2-induced K-562 cells. The circRNA.0007127—miR-513a-5p and CASP8—miR-513a-5p interactions were verified by luciferase reporter assays. Results: Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells. Compared with the control group, the expression of CASP8 was reduced by 50% and the 43-kD fragment of CASP8 protein was significantly reduced (P⩽0.05). The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8, with extremely significant differences (P⩽0.001). The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model (75% change) and the level of a 43-kD fragment of CASP8 protein (P⩽0.01). The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA (siRNA) and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist. Conclusions: CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis, which can serve as a novel powerful molecular target for K-562 cells.
Abstract
目的
探讨CCR4-NOT复合物亚基2基因(CNOT2)来源的环状RNA circRNA.0007127在凋亡通路中的作用。
创新点
本研究发现,circRNA.0007127与K-562细胞凋亡相关,可能是一个预测K-562细胞凋亡的分子标志物。并首次证实了细胞凋亡中circRNA.0007127/miR-513a-5p/CASP8的调控轴。
方法
使用miRanda、TargetScan和RNAhybrid软件对circRNA.0007127的靶微小RNA(miRNA)进行预测,筛选具有凋亡相关基因结合位点的miRNA。在H2O2诱导的K-562细胞中,通过定量聚合酶链反应、流式细胞术、线粒体膜电位、免疫荧光、蛋白印迹和胱天蛋白酶8(CASP8)蛋白活性实验来对circRNA.0007127及其下游靶点miR-513a-5p进行体外验证。circRNA.0007127–miR-513a-5p以及CASP8–miR-513a-5p的相互作用分别通过荧光素酶报告基因检测得到验证。
结果
沉默circRNA.0007127通过抑制CASP8通路激活降低K-562细胞凋亡。与对照组相比,CASP8表达减少2倍,CASP8蛋白43-kD片段显著减少(P<0.05)。荧光素酶报告实验表明,circRNA.0007127可以结合miR-513a-5p或CASP8,差异极显著(P<0.001)。miR-513a-5p过表达抑制K-562细胞中CASP8基因表达水平(4倍)和CASP8蛋白43-kD片段水平(P<0.01)。挽救实验表明,circRNA.0007127 siRNA和miR-513a-5p抑制剂共转染增加CASP8基因表达和凋亡率,提示miR-513a-5p抑制剂是circRNA.0007127的拮抗分子。
结论
CircRNA.0007127通过miR-513a-5p/CASP8轴调控K-562细胞凋亡,可作为K-562细胞新的分子靶点。
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Acknowledgments
This work was supported by the Guangzhou Science and Technology Plan Project (No. 201904010027) and the Key Clinical Technology Program of Guangzhou (No. 2019ZD18), China.
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Xiajing LI designed and conducted experiments, obtained and analyzed data, and was a major contributor in writing the manuscript. Yiyu ZHANG provided sequencing data and experimental design. Ning WANG conducted experiments and analyzed the data. Zhaohu YUAN provided information support, and analyzed and interpreted the data. Xiaojie CHEN collected and processed experimental samples. Qicong CHEN conducted experiments. Hui DENG and Xinxin TONG collected experimental samples and performed statistical analysis. Honglin CHEN participated in paper revision and performed statistical analysis. Yuyou DUAN provided experimental design, performed statistical analysis, analyzed and interpreted the data, and participated in paper revision. Yaming WEI provided experimental design and financial support, conceptualized the outline and topic of the article, and participated in paper revision. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Xiajing LI, Yiyu ZHANG, Ning WANG, Zhaohu YUAN, Xiaojie CHEN, Qicong CHEN, Hui DENG, Xinxin TONG, Honglin CHEN, Yuyou DUAN, and Yaming WEI declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Li, X., Zhang, Y., Wang, N. et al. CircRNA.0007127 triggers apoptosis through the miR-513a-5p/CASP8 axis in K-562 cells. J. Zhejiang Univ. Sci. B 23, 732–746 (2022). https://doi.org/10.1631/jzus.B2200048
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DOI: https://doi.org/10.1631/jzus.B2200048